Systematically Engineering for Efficient Production of 3‐Methyl‐1‐Butanol in Escherichia coli

3-Methyl-1-butanol (3-MB), a promising next-generation biofuel, has garnered significant interest owing to its superior combustion characteristics and fuel compatibility. However, current 3-MB biosynthesis faces major challenges, including low production efficiency and severe toxicity-induced growth inhibition, which significantly limit its industrial feasibility. In this study, we systematically developed an integrated metabolic engineering approach for high-level 3-MB production in Escherichia coli. Through semi-rational engineering of the rate-limiting enzyme dihydroxyacid dehydratase (DHAD), combined with molecular dynamics simulations, we identified and addressed previously unrecognized catalytic bottlenecks. The engineered strain exhibited a 32.3-fold increase in 3-MB production, reaching 2.20 g/L in shake-flask cultures. Subsequent adaptive laboratory evolution further improved strain robustness, while genomic analysis revealed novel regulatory targets for metabolic optimization. In a scaled-up bioreactor fermentation system, the final strain achieved a record titer of 6.24 g/L, representing the highest reported titer for engineered microbial systems. This work not only establishes a scalable platform for 3-MB biosynthesis but also provides a modular engineering framework applicable to other advanced biofuels.